Biochimie (1991) 73, 1261-1268

© Soci6t6 fran~aise de biochimie et biologie mol6culaiie / Elsevier, Paris

1261

Review

The remote control of transcription, DNA looping and DNA compaction M Amouyal Unit~ de physicochimie des macromol~cules biologiques, d~partement de biologie mol~culaire, 25, rue du Docteur Roux, 75724 Paris Cedex 15, Francc

(Received 7 May 1991; accepted 19 September 1991)

Summary - - mRNA synthesis can be controlled at some distance from the start of transcription in eukaryotes and prokaryotes. It is generally assumed that the distal elements of the transcriptional machinery directly interact with the proximal elements, forcing the DNA to bend in a loop. DNA loop formation and transcription can be affected by the distance between the sites, their helical positioning, their orientation, their concentration (responsible for a cis- or a trans-effect of the DNA sequences), and DNA compaction in chromatin. The corresponding in vitro and in vivo situations have been critically examined for a number of systems, mostly prokaryotic. regulation of transcription / enhancers / repression / DNA looping / sliding / chromatin / DNA compaction / distant regulation / DNA-protein interactions

D N A loop formation was first considered for repression of the E coli lactose operon [ 1]. This model was based on physicochemical considerations. The repressor existing at a low copy number in the cell was supposed to be trapped by the large amount of nonspecific DNA. Then, natural folding of D N A ( D N A looping) helped to transfer the protein to it specific site, the lac operator O 1. This operator is located close to the start of transcription. The discovery of remote operators by sequence homology with O 1 and in vitro assays (see for example [2]) and the finding that the tetrameric lac repressor needs only two subunits to recognize the operator in vitro ',ed to a variant of this view. The protein was now supposed to be stably chelated to the proximal and distant operators in the loop so that dissociation from the proximal operator O1 was prevented [3]. But in a first analysis the constitutive mutations for the lac operon were all f o u n d located in the first operator region and repression at a distance as well as D N A looping were prematurely discarded (for additional historic details about lac repression at a distance, see review [4]). The contribution of remote elements, known as enhancers, to transcription and other biological processes was really discovered in eukaryotes, in the early 80s, without any reference to D N A looping.

Enhancers are specific DNA sequences and the corresponding DNA-binding proteins. When involved in transcription, they are responsible for the high (or low) level of gene expression when the proximal elements of the transcription machinery initiate (or repress) transcription poorly by themselves. They can function over considerable distances from the site of initiation, in the normal or inverted orientation [5-7]. The first prokaryotic counterparts were found soon afterwards in the galactose and a r a b i n o s e operons and DNA looping was again proposed to explain the observed long range effects on repression of these and other operons (for reviews about prokaryotic enhancers, see [8-10] and for a critical evaluation of the various modes of remote control in prokaryotes and eukaryotes, see [11]). Paradoxically, with regard to the long distance effects stressed above, the need to prove the direct interaction between two proteins normally binding to adjacent sites also led to D N A looping. Upon separation of the sites, D N A flexibility was incidentally found to be rr:sponsible fer the cooperativity of binding of phage 2~,cl repressors, in addition to protein flexibility previously considered [ 12]. Furthermore, an important step for D N A looping was provided by the works on the spontaneous cycli-

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I ~

M Amouyal

zation of a DNA fragment [13, 14]. The corresponding knowledge of the factors which affect this process influenced more or less strongly several studies. Mainly, the dependence of DNA cyclization on the distance was deliberately used to prove DNA loop formation in vivo for the first time in a lac system [ 15]. Independently, the extensive translation of the cyclization studies into terms of DNA looping allowed to predict some of the conditions necessary for protem-DNA loop formation in vitro, when these structures were only postulated [16]. Moreover, the use of protein-DNA loop~ as models for DNA circles lead to in vitro and in vivo biophysical applications [ 17, 18] - w h i c h will not be developed in this review restricted to the functional aspects of DNA looping. In the first section of this paper, the analogy with DNA cyclization is used as a guideline to present some of the requirements for protein-DNA loop formation in vitro, such as the distance between the sites, their helical positioning, orientation and concentration, with implicit reference to the known properties of enhancers and new developments (first and also sect~ad section). In the following sections, some of the recent information about their influence on the remote control of transcription is examined with emphasis on prokaryotic studies. The last section discusses more specifically the possible occurrence of other modes of remote control.

Direction of a protein-DNA loop This section is restricted to the works which illustrate closely some of the structural requirements for DNA loop formation observed with purified components. The E coli lac operator-repressor loop is a well documented system from this point of view ([4], and this review). It is used here as a convenient presentation aid. DNA cyclization and DNA loop formation are ruled by DNA flexibili~

For DNA cyclization this was indicated by the existence of an optimal leagth of the fragment around 400 bp [ 13, 13a]. When the distance between two lac operators was close to this value, 535 bp precisely, electron microscopy revealed entire fields of DNA loops whereas below [16] or above (1873 bp) (M Amouyal, unpublished observation), only onethird to half of the proportion of the 535-bp-loops was observed with the constructions that we employed. This is the only known observation of an optimal distance for DNA protein-loop formation. This maximum which really correlates protein-DNA looping to DNA cyclization indicated that DNA flexibility is inherent in the process of protein-DNA looping.

Loop formation can occur even when ring closure is generally not possible, below 150 bp. For these short distances, DNA only needs to bend and not to circularize for the protein to contact simultaneously both sites, as first reported for 2~,cl repressor (52- and 63-bp loops [12, 19]). It was also true for lac repressor (63- to 147-bp loops [16]). Therefore, because of DNA flexibility: DNA loops can form on a whole range of distances, from a few ten base pairs to several thousand base pairs. Additionally to the lac case already detailed, long distance DNA looping has been directly observed for the E coli R6K plasmid replication enhancer-origin interaction [20], the Hininversion system of Salmonella [21], the E coli RepA protein and mini-P1 plasmid replication [22] and E coli type II endonuclease Nael [23]. Intermediate distances have been observed with the bacterial transcriptional activator NRI contacting the t#4-containing RNA polymerase [24], the DeoR repressor of the E coli deo operon [25] and for the association of DNA-bound progesterone receptors [26]; as regards DNA looping, DNA is considered as a flexible thread without any biological content which obeys the theory of flexibility of polymers [27]. As a result, it is indifferent to any modification, even a partial replacement of DNA [28], as long as the 'thread' does not become rigid. This property has been used to discriminate DNA looping from other mechanisms (see last section). Specific sequences or proteins inducing DNA bending are not required for DNA loop formation, except when DNA becomes rigid, ie for short distances. In this case, the sequence, the structural defects of DNA or the proteins which ~ _ _, _ . m o u , y DNA .neXlolmy ...... between the sites of interest may favor (or disfavor) DNA loop formation. Thus a site in a 5 bp TTTAT sequence between the two lac operators O1 and 0 3 is hypersensitive to the attack of various chemical probes in the presence of negative supercoiling [29]. According to the authors, this sequence becomes particularly susceptible to DNA melting and bending when submitted to torsional stress. Several biological processes require the assembly (or disassembly)of nucleoproteic structures [30], and DNA bending assists DNA looping in some of these processes. The ~, phage integrase protein (lnt) is one of the proteins responsible for integrative recombination between the phage and bacterial sites and for excisive recombination between phage sites. The bacterial integration host factor IHF promotes the simultaneous binding of lnt to its sites by bending the DNA in the middle of the 50 bp separating the sites [31]. A naturally bent DNA sequence (an 'A-tract') can replace successfully the IHF binding site [32]. The catabolite activator protein (CAP), known to bend DNA, could replace IHF with the same effect [32].

DNA looping for transcription in prokaryotes and eukaryotes

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IHF also stimulates the interaction of o54-RNA polymerase with NifA, the transcriptional activator for nitrogen fixation operons of Klebsiella pneumoniae [33, 34]. There are also some indications that IHF helps the assembly of proteins at the origin of pSC 101 plasmid replication [35]. Similar functions have been proposed for the E coli histone-like protein Hu [35] and CAP [J 7, 32, 37]. The formation of a nucleoproteic assembly between E coli gal repressor and RNA polymerase would lead to gal repression [37, 38].

DNA cyclization is also ruled by DNA torsional rigidity In other words, the two extremities of a short DNA fragment have to be exactly in phase, ie separated by an integral number of helical turns /'or covalent joining. However, due to additivity of the elementary thermal fluctuations of DNA torsion at the level of each base pair, this phasing requirement is progressively lost when the size of the fragment is increased [39]. When ring closure is mediated by proteins, the proteins may affect the structure of the loop. Contrary to DNA cyclization, the two sites do not have to be phased on DNA to ensure the specific contacts. But if multiples of helical turns are introduced between the two sites, their initial orientation will be restored. Thus, NtrC and oS4-RNA polymerase directly interact, though lying on different faces of DNA [41, 42]. For lac repressor, the two sites had to be in phase to induce stable loop formation, as evidenced by gel retardation assays [ 16]. It is generally not emphasized UlgI, L t i l e , ' ~ I L C b U l l the p~otcin are not fiecessa parallel, though this may be crucial for the analysis of the data as in [ 18], or simply for a better knowledge of the structure of the protein. Lac repressor is composed of four identical subunits and binds as a dimer [3]. The arrangement of the subunits is not known unequivocally, due partly to the poor crystallisation of lac repressor and lack of high resolution X-ray diffraction studies [43]. A parallel arrangement of the two dimers is generally postulated, as in [44], but a tetraedral arrangement has also been proposed (reviewed in [45]). When the two phased operators were included in a relaxed mini-circle, only the configuration drawn in figure 1a was observed by electron microscopy (see fig 2b of [18]) indicating a parallel arrangement. A tetraedral organization would have led to an eight configuration of the circle (fig 2b). The periodic formation of stable loops results from stiffness of both DNA and protein, a helical pitch for DNA with a given value, and a unique configuration of the loop. Dissociation assays of preformed complexes or association assays in the presence of IPTG displayed an apparent lack of phasing require-

(ct)

(b3

Fig 1. Protein architecture and loop formation on a relaxed circle: the contacts on the protein ai-e parallel (a), orthogonal (b).

ment in that stable loops were formed at any distance between 158 and 168 bp on highly supercoiled DNA with lac repressor [16]. For these same distances on the fragment, DNA loops were also formed when the two operator sites were not in phase, because of the amplitude of the natural movement of torsion and detorsion. However, they were less stable, as indicated by the smeary aspect and weak density of the band assigned to the complex. DNA supercoiling amplifies this phenomenon, since it naturally brings closer the different parts of DNA. It also changes the helical pitch of DNA and the distances, previously inappropriate for alignment of the sites on the fragment, Lrt...t,.,IL]iliI,,., ~Ultl~l,I..#lli~ ~ILTII 3UI.#Ig~II,,..UII~ILI L..Jl~ll"l_ L I 0 ] .

Protein DNA loop formation with asymmetric sites In large loops such as the 535-bp lac loops previously mentioned, DNA can adopt the two configurations (a) and (e) drawn in figure 2. Below 150 bp, only one configuration (d), equivalent to (a), was observed (see figs 7 and 8 of [16]). Both lac repressor and the 'ideal' lac operator used in these experiments are perfectly symmetrical. If they were not symmetrical, the existence of two configurations when DNA has lost its torsional and flexional rigidities, clearly shows that DNA looping is insensitive to the orientation of the site at these distances but might sense it over short distances. For this to be true, the asymmetry at the level of the binding site must be associated with asymmetry at the level of the protein-protein contacts. Enhancer proteins are often homodimers with a two-fold symmetly when they are free in the solution. However, the protein might deviate from symmetry once bound to its site.

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M Amouyal

(a)

(b)

(el

(d)

(el

If)

Fig 2. Site inversion and loop formation: the arrow indicates the coding sequence. The asymmetry of the site is supposed to be transmitted to the protein. Consequently, the orientation of the site with respect to the coding sequence is indicated by the asymmetrical shape of the protein. In this example, a loop is formed when the two sites have the same orientatien (a and d). When they are in the opposite direction, the two configurations of DNA (b) and (c) are possible for long distances and a loop. can form (c). At short distances, only one D N A configuranon is possible (e) and

the loop cannot form (f).

DNA cvclization depends on D N A concentration

The cyclization probability or j-factor represents in fact the DNA concentration such that cyclization and dimerization of the fragment are equally probable [ 13, 14]. The scheme of figure 3 translates this situation into terms of DNA looping for the lac system. At low concentrations of DNA and repressor, the intramolecular reaction for DNA, ie DNA looping, is favored over the intermolecular reactions and the formation of 'sandwich'-type species. 'Tandems' are formed in medium concentrations. All these structures have been evidenced by gel retardation and em assays, on this basis [ 16].

Remote control of transcription Distances

Originally noticed for their ability to function at a distance, enhancers now frequently appear to operate both from a few ten to several thousands of bases. Some examples of such a performance among prokaryotes are provided by the nitrogen regulatory protein )

----,---'~-'---.]~-

-

"loop"

F~.

"tandem"

"sandwich

"

Fig 3. Influence of DNA and protein concentrations on DNA loop formation, with lac repressor as the protein.

NRI (also called NtrC or GInG, for reviews about this transcriptional activator, see [46, 46a]), which operates from 30 to 1400 bp upstream and -- 2000 bp downstream [42, 47], the nitrogen fixation protein NifA (= 100 to 2150 bp [48]), the DeoR repressor (66 to 4600 bp [49]), the Hin enhancer for site specific inversion and recombination (-- 100 to 4000 bp [50]) and to a lesser extent, by the AraC protein involved in repression of the araBAD operon (32 to 500 bp [51]). The low affinity of the araC protein for one of its sites limits the range of operational distances [51 ]. When both short and long distance effects have been observed, a feature already consistent with DNA loop formation, there is generally some more direct evidence for DNA looping. Loops could be visualized by electron microscopy for NRI [25], DeoR [25] and Hin [21]. They were detected electrophoretically in case of araC [52]. NifA has not been purified yet. However, it is closely related to NRI. Like NRI, it functions with oS4-containing holoenzyme rather than oT0-holoenzyme [53, 54]. The protein synthesized in vitro is required for activation of a NifH-lacZ gene fusion [53]. Chemical 'footprinting' shows that the proposed distant sites are occupied in vivo [55] and that like NRI, its presence is required for open complex formation at the promoter in the cell [56, 57]. Like for some of the above-mentioned enhancers, indirect evidence for DNA looping exists [33] that will be presented later. Prokaryotic enhancers have generally lost the major part of their power above 5 kbp (see [58] for DeoR, [48] for NifA, [51] for AraC, [59] for TyrR repressor and distant repression of the E coli aroF promoter, [60] for lac repressor). Above this same value, ring closure probability is also very weak, just as low as below 150 bp under the conditions defined by Shore et al [ 13]. An important consequence is that prokaryotic DNA is not strikingly different from 'naked' linear DNA in solution. Furthermore, regulation of gene expression from distant sites is much more frequent in eukaryotes than in prokaryotes [61 ]. Thus, in E coli, the few examples of repression or activation at a distance with a natural location of the distant site from 90 to 900 bp, would constitute exceptions. On the contrary, in higher eukaryotes, a typical promoter transcribed by RNA polymerase II would include an enhancer sequence located or functioning 'at least up to 10 kbp from the RNA start' [6, 62]. In eukaryotes, the chromatin is highly organized into nucleosomes and higher order structures, while DNA packaging in bacteria is considered more labile, though not much is known [63]. The true distance of action of an eukaryotic enhancer may then be shorter than that calculated from naked DNA. Along these same lines, the distance effect should reflect the differences in DNA condensation between

DNA looping for transcription in prokaryotes and eukaryotes eukaryotes and prokaryotes and, for me, will constitute a tool to detect such differences. It might be related to this point that some mammalian enhancers which work over a few kb in vivo cannot work over 500 bp in vitro, in the absence of chromatin [28].

1265

operators instead of the wild type ones had probably the same effect, as stressed in [ 18]. The wt operators indeed form less stable loops than the 'ideal' ones in vitro under the same experimental conditions [70]. A fortunate consequence is that full inducibility of the lac operon can be ensured 1181.

Helical orientation of the enhancer sequence Orientation with respect to the coding sequence

Stimulation sometimes depends on the correct helical positioning of the enhancer with respect to the proximal elements. This is revealed by insertions (or deletions) of successively odd and even numbers of helical turns between the sites of interest, at reasonably short distances, as expected if DNA acquires some torsional stiffness (see for example [17, 33, 42, 51, 64-66]). An extreme situation is that of adjacent proteins such as the ;Z phage repressors [ 12]. It is generally not stressed that a periodic stimulation is not per s e a proof for a direct cooperative interaction between the proteins of interest. Mediation by another protein [21, 67], DNA conformational changes at a distance or the 'sandwiches' of figure 3 may exhibit tbe same property. In some cases, on the contrary, there is some evidence in favor of DNA looping while there is no or poor helical periodicity for enhancer function. (It is assumed that all the length of the spac~.r between 0 and 10 bp has been explored, since the interaction between the two proteins may not be optimal in the wild type situation.) Thus, the DeoR repressor has three or four DNA binding sites and a loop might be formed for different helical positions of the sites [25, 49]. Only weak periodicity was observed for lac repression In viva., ,.,,.,, ,,, ,_,.., ,u o,, or, ,_,~tw~,~ a,, 'ideal' lac operator and an operator with a constitutive mutation (H Kriimer, PhD thesis, 1988, Cologne, Germany). DNA is supercoiled in bacteria and the situation observed in vitro [18] might be reproduced in vivo. Similarly, a strict alignment is not required in vivo between the Gal4 activator and the 'TATA' box elements of the yeast galactokinase gene [68]. This is also true for some other systems quoted in this work (see also [69] for a recent example). According to the authors, an especially strong interaction between Gal4 protein and RNA polymerase II, forcing D N A to twist, might be responsible for this situation, as well as the flexibility of the interacting proteins (see also [65]). The degree of periodic stimulation also depends on the affinity of the enhancer protein for its site. A strong interaction in conjunction with the thermal fluctuations of the twist likely stabilize DNA looping at any distance. Such a reduced effect of phasing was observed in vivo when a high NRI binding site replaced a low affinity one [42]. The use of 'ideal' lac

Sequence inversion is generally performed at the same time as the site is moved away from its natural location. It is found that inversion has nearly no effect on stimulation (numerous examples are given in reviews [5, 7, 62]). Influence of sequence inversion on expression over short distances is less documented. The scheme of figure 2 suggests that the bidirectional stimulation is not necessarily maintained over short distances if the enhancer sequence is part of a loop. This is in principle a way to recognize DNA looping from other mechanisms in vivo (M Amouyal and B von Wilcken-Bergmann, work in progress). Intracellular concentrations of enhancer DNA and protein

These concentrations classically vary for technical reasons (eg multi-copy plasmids or overproduction of the protein) or in response to environmental changes, as in case of nitrogen deprivation for NRI [46, 46a]. A gradient of concentration of regulatory proteins can also be produced naturally as reported for Drosophila development [71]. These concentrations can also, to some extent, be used as a physicochemical parameter 41~---- ~ - k - - - - - - ! ! IUI U l I ~ t.~K;II.

High amounts of DeoR repressor have the same effect as distant sites for repression in the deo operon [72]. Thus, the remote operators indirectly increase the local concentration of repressor, a function of the loop stressed by Record and collaborators [ 15]. The cooperativity of repression observed between the three lac operators is lost with a dimeric repressor unable to aggregate into the tetrameric form and to induce DNA loop formation in the wild-type situation [73]. This result definitively stopped the controversy about the involvement of the distant operator sites in repression of the lac operon (Introduction). It could be obtained because the dimeric repressor was deliberately used in combination with the low chromosomal concentrations of repressor and DNA, contrary to previous in vivo works. With higher concentrations of repressor (iqI strains), the cooperativity between the three operator sites was greatly reduced, and no difference in the level of repression between the tetrameric and the dimeric repressor was observed. Therefore, the high amounts of repressor had induced the predicted in vivo transition from DNA looping to another

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M Amouyal

mode of repression (previous section and [16]) and the three lac operators were now separately occupied ('tandem" formation). Concentrations are also critical for the cis/trans effect of enhancer sequences. In physicochemical terms, this situation covers intra- and intermolecular reactions for DNA. Some mechanisms such as sliding imply that the enhancer functions exclusively in a cisposition. On the contrary, DNA looping, normally cisacting, can be compensated in trans. Up to now, the trans-effect of transcriptional enhancers has been exclusively observed when the two sites of interest w e n maintained in close proximity, in vitro, with catenanes [74, 75] or a non-covalent bridge [28], and in vivo, in the natural phenomena of transvection in Drosophila [76]. The question has been raised of whether it would be possible to detect allelic interaction of the transvection type in organisms where there is no apparent somatic pairing of chromosomes [76]. A trans-interaction between two sites carried by two unlinked DNA molecules should be observed by trans-complementation with high amounts of DNA in the cell, in order to form 'sandwich'-type structures. The NifA aperiodic activation of NifH promoter on multi-copy plasmids is probably an example of such a trans-effect [33]. Along the same lines, activation of the prokaryotic glnA and Nifl-I genes is also possible in the absence of enhancer site and the presence of high concentrations of the corresponding proteins NRI [47, 74] and NifA [77]. The future will tell if equivalent situations can naturally occur. Redundancy of information

Enhancer sequences are generally repeated [5, 62]. They may allow cooperative interactions between the corresponding proteins and facilitate the contact between the erdaancer protein and the components of the transcriptional machinery at the promoter. This is how the ;L cl repressors stimulate repression from the P,m promoter [12] and how NRI would also activate expression [42]. The enhancer proteins may also cooperate not by directly touching but by simultaneously touching some component of the transcriptional machinery [78, 79] or by aggregating ([5] and references therein). Repeated sites can also simply increase the probability of single loop formation at one site or favor the formation of multiple loops, reinforcing all the functions of single loops, as observed for the deo operon [25].

enhancer activity in trans was observed in case of a total or partial disruption of the.DNA track [28, 75]. Em did not reveal several (unspecific) locations of lac and deo repressor on a fragment (see all em pictures in [16] and [25]), though for lac repressor, the high affinity 'ideal' lac operators were used. Such experiments rule out sliding as exclusively acting under the precise in vitro conditions urder which they were performed. In vivo, the use of psoralen-modified DNA between the SV40 enhancer and the human I~-globin gene inhibited expression of this gene [81 ]. But psoralen alters DNA properties ([28] and references therein). On the contrary, NifA activation was not inhibited by lac repressor binding in the middle of DNA sequence between upstream and downstream elements in vivo [33]. Also, loss of cooperativity in vivo between distant and proximal sites with proteins engineered so that they become unable to aggregate [73, 82, 83] directly supports the existence of DNA looping in the cell. This is especially true when the loop involves a unique and multimeric protein such as lac repressor, since otherwise, the question of possible intermediate factors mediating the protein-protein contacts is raised in the absence of an in vitro proof for direct interaction. Yet, sliding might assist DNA looping. Recent biophysical experiments suggest it might be the case for lac repressor and wt operators [84]. A distantly bound protein can also block RNA elongation [66, 85, 86]. The enhancer can also interfere with other regulatory proteins and other processes at the same location: the repressor hinders CRP activator binding when located upstream from the tran. . . . Fuu,l start in tuc e L'U 11 gatactose operon [40, 87], while there is no in vitro evidence for DNA looping with this repressor [37]. However, it presumably exists in vivo. Indeed, a gal to lac operator conversion in vivo allows efficient repression of the gal operon [88], and this repression is relieved when a dimeric lac repressor is used [38]. Furthermore, various modes Gf remote control can be assumed by DNA looping, as stressed by Flashner and Gralla [85], in case of blockage of RNA elongation. Finally, in somes cases, environmental changes induce the transition from DNA looping to another mechanism. Variations of concentrations lead to such a situation [16, 73]. Variations of DNA superhelicity, due to an osmotic shock or anaerobiosis ([89] and references therein) or DNA transcription [90-92], might also favor such transitions with low affinity operators to ensure loop breakage [ 18].

The other modes of remote control

Trapping of the protein by the distant site and sliding to the site of interest is often viewed as the main alternative to DNA looping [5, 80]. But retention of

Acknowledgment David Perrin is acknowledged for careful reading of the present article in 1990.

DNA looping for transcription in prokaryotes and eukaryotes

References

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